Pharmacogenetics is the study of the role of inheritance in variation in drug response phenotypes. The pharmacogenetic research program supported by this grant has focused on the pharmacogenetics of Phase II (conjugation) reactions, particularly methylation. Methylation plays an important role in the metabolism of many drugs, neurotransmitters and hormones as well as critical cellular processes such as the CpG methylation of DNA. During the present funding cycle, our studies of methylation moved beyond the pharmacogenetics of the methyltransferase (MT) enzymes themselves to include studies of genetic variation in genes encoding enzymes in the Methionine and Folate Cycles that generate the methyl donors for MT enzymes, S-adenosyl-L-methionine (AdoMet) and methyltetrahydrofolate, CH3THF. During that same time, we also performed preliminary pharmacogenetic and pharmacometabolomic studies of clinical response to selective serotonin reuptake inhibitors (SSRIs), antidepressant drugs that alter the function of the monoamine neurotransmitters that we have studied extensively because of the important role of methylation in their biosynthesis and metabolism. The results of those SSRI pharmacometabolomic experiments, in concert with the results of metabolomic studies of SSRIs performed by others, indicated that methylation and the Methionine and Folate Cycles may play an important role in variation in SSRI clinical response. Therefore, in the present application, we propose to build on preliminary results obtained during the present funding cycle to perform a comprehensive, integrated series of studies of the pharmacogenetics of the pathways that generate methyl donors, utilizing metabolomic assays of small molecule intermediates in the Methionine and Folate Cycles performed with preparations from a genomic data-rich cell line model system that has already demonstrated its power and utility for generating and testing pharmacogenomic hypotheses. We will also perform complementary studies using a large number of clinical hepatic biopsy samples. We have already used a similar approach successfully in preliminary studies performed during the present funding cycle. In parallel with these laboratory-based experiments, clinical pharmacometabolomic studies of SSRI response will be performed, focusing on the contribution of the Methionine and Folate Cycles to individual variation in SSRI outcomes. These translational studies are possible because of a large, ongoing clinical trial of SSRI drugs that is being performed at the Mayo Clinic. The results of this series of integrated experiments will significantly increase our understanding of the pharmacogenomics of methylation and will also utilize pharmacometabolomics to inform pharmacogenomics-in this case the pharmacogenomics of methyl conjugation as well as the contribution of pharmacogenomic and pharmacometabolomic variation to individual differences in clinical response to SSRI therapy.